The Carrot Phytoene Synthase 2 (DcPSY2) Promotes Salt Stress Tolerance through a Positive Regulation of Abscisic Acid and Abiotic-Related Genes in Nicotiana tabacum

Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines.

In higher plants, carotenoid biosynthesis takes place in plastids via the methylerythritol phosphate (MEP) pathway and begins with the generation of 15-cis-phytoene from two molecules of geranylgeranyl pyrophosphate (GGPP), a process catalyzed by phytoene synthase (PSY). This step represents both the first committed stage and a crucial point for regulating the flow of carotenoids ( Figure 1) [10][11][12][13]. Subsequently, 15-cis-phytoene is transformed into di-cis-ζ-carotene by two subsequent desaturation reactions, catalyzed by phytoene desaturase (PDS) and 15-cis-ζ-carotene isomerase (Z-ISO) and then desaturated by ζ-carotene desaturase (ZDS) and carotene isomerase (CRTISO), producing all-translycopene. Next, all-trans-lycopene is converted to different bicyclical molecules. The enzyme lycopene β-cyclase (LCYB) turns all-trans-lycopene into β-carotene, and both LCYB and lycopene ε-cyclase (LCYE) give raise to α-carotene. β-carotene is the substrate of carotene β-hydroxylase-1 (CβHx) needed to produce zeaxanthin, whereas the hydroxylation of α-carotene by two heme-containing cytochrome P450 enzymes (CYP97A3 and CYP97C1) and CβHx produces the yellowish carotenoid lutein. Zeaxanthin is turned into violaxanthin by the action of zeaxanthin epoxidase (ZEP) (and VDE converts violaxanthin into zeaxanthin). After several enzymatic reactions, ABA is produced in the cytoplasm. The initial stage of ABA biosynthesis, which is a crucial step in the production of this hormone, is catalyzed by NCEDs. This makes NCEDs a bottleneck in the biosynthesis process. In higher plants, carotenoid biosynthesis takes place in plastids via the methylerythritol phosphate (MEP) pathway and begins with the generation of 15-cis-phytoene from two molecules of geranylgeranyl pyrophosphate (GGPP), a process catalyzed by phytoene synthase (PSY). This step represents both the first committed stage and a crucial point for regulating the flow of carotenoids ( Figure 1) [10][11][12][13]. Subsequently, 15-cis-phytoene is transformed into di-cis-ζ-carotene by two subsequent desaturation reactions, catalyzed by phytoene desaturase (PDS) and 15-cis-ζ-carotene isomerase (Z-ISO) and then desaturated by ζ-carotene desaturase (ZDS) and carotene isomerase (CRTISO), producing all-translycopene. Next, all-trans-lycopene is converted to different bicyclical molecules. The enzyme lycopene β-cyclase (LCYB) turns all-trans-lycopene into β-carotene, and both LCYB and lycopene ε-cyclase (LCYE) give raise to α-carotene. β-carotene is the substrate of carotene β-hydroxylase-1 (CβHx) needed to produce zeaxanthin, whereas the hydroxylation of α-carotene by two heme-containing cytochrome P450 enzymes (CYP97A3 and CYP97C1) and CβHx produces the yellowish carotenoid lutein. Zeaxanthin is turned into violaxanthin by the action of zeaxanthin epoxidase (ZEP) (and VDE converts violaxanthin into zeaxanthin). After several enzymatic reactions, ABA is produced in the cytoplasm. The initial stage of ABA biosynthesis, which is a crucial step in the production of this hormone, is catalyzed by NCEDs. This makes NCEDs a bottleneck in the biosynthesis process [14][15][16][17][18][19] (Figure 1). ABA participates in many physiological processes including seed dormancy, Plants 2023, 12,1925 3 of 17 regulation of plant growth, senescence and control of stomatal aperture, and in tolerance to abiotic stresses such as cold, drought and salinity [14][15][16]18,20].
PSY has been functionally characterized in different plant species, such as Solanum lycopersicum (tomato) [14,21], Arabidopsis thaliana [22], Zea mays (maize) [23], Cucumis melo (melon) [15,24], Narcissus pseudonarcissus (narcissus) [25] and Capsicum annuum (pepper) [26], among other species. Whereas PSY is present in a single copy in Arabidopsis, paralogous genes have been identified in several economically important crops, such as tomato, maize, tobacco, Oryza sativa (rice), melon and Manihot esculenta (cassava) [11,14,[27][28][29]. In tomato, rice and maize, three paralogous PSYs with different roles have been described [11,29]. In tomato, SlPSY1 is expressed mainly in fruits, whereas SlPSY2 is more prevalent in leaves and flowers [14,30,31]. SlPSY3 is predicted to respond to abiotic stress, like PSY3 from rice and maize that responds also to ABA treatments [11,29]. On the other hand, PSY1 and PSY2 of these cereals are induced by light in leaves [11,29,32]. Therefore, the available evidence indicates that there is a range of functional specificity and diversity among PSYs in plants. The expression of PSY in the root of Arabidopsis increases carotenoid accumulation in response to osmotic and salt stress, as well as the external application of ABA, according to studies by Meier et al. (2011) and Ruiz-Sola et al. (2014) [33,34]. This indicates that ABA controls the production of its own metabolic precursors.
In carrot, one of the few plant species that synthesize and accumulate carotenoids in the storage root in the absence of light, DcPSY1 and DcPSY2 paralogs have been identified [35,36]. Both genes present differential expression patterns during leaf and root development. The expression of DcPSY1 is specifically triggered in leaves, which supports its function in the synthesis of carotenoids in chloroplasts [37,38]. On the other hand, DcPSY2 exhibited elevated expression levels during storage root development, which is characterized by a marked rise in the overall carotenoid levels [37,38]. Moreover, the expression of DcPSY2 correlates better than DcPSY1 with the accumulation of carotenoids in roots of the orange cultivar with respect to wild white (Ws) carrot inbred lines [39]. Arias et al. (2021) found that overexpression of DcPSY2 in tomato and, transiently, in Malus domestica (apple) fruits resulted in a significant increase in total carotenoids, including β-carotene, which was consistent with the expected subcellular localization of DcPSY2:GFP in the plastids.
The results obtained from this study suggest that DcPSY2 is a functional gene that holds great potential to enhance the carotenoid content in fruits of various commercial species. Furthermore, it was observed that the expression of DcPSY2 and DcPSY1 is significantly upregulated in response to salt stress in both the leaves and roots of carrot seedlings, and this upregulation is positively correlated with an increase in ABA levels (Simpson et al., 2018). However, only DcPSY2 expression is induced by ABA in carrot roots, due to the presence of ABREs elements in its promoter [40]. To further investigate the potential benefits of DcPSY2, we conducted experiments by overexpressing this gene in Nicotiana tabacum (tobacco) and assessing its impact on salt stress tolerance. Our findings showed that DcPSY2 T 1 transgenic tobacco lines exhibit an increased survival rate after salt treatment, and this effect is directly associated with the upregulation of abiotic-related gene expression and ABA levels in roots. These results complement and expand on previous research in this area.

Vector Construction
For DcPSY2 expression in N. tabacum, the corresponding coding sequence of 1276 bp was amplified from carrot storage root by RT-PCR with Pfu polimerase (Promega, Madison, WI, USA) and Psy2F and Psy2R primers (Supplementary Table S1) and cloned into pCR8 ® /GW/TOPO vector (Invitrogen ® , Waltham, MA, USA) according to the manufacturer's indications. Positive clones were selected by PCR, enzymatic digestion and sequencing (Macrogen Corp, Rockville, MD, USA, Supplemental Figure S1). Subsequently, pCR8/DcPSY2 positive clones were recombined into the binary vector pGWB2 [42], to generate pGWB2-DcPSY2, then introduced into Agrobacterium tumefaciens (GV3101 strain) for stable DcPSY2 expression in tobacco. Real time RT-PCR was performed with tree biological replicates measured twice each. Ubiquitin gene expression was used as normalizer. As calibrator, DcPSY1 expression in 4-week-old leaves (L 4W) and DcPSY1 in 4-week-old roots (R 4W) were used and settled in 1. Error bars represent standard error of the mean (three technical replicates; asterisks indicate Student's t-test significant differences * p < 0.05, ** p < 0.01, *** p < 0.001).

Vector Construction
For DcPSY2 expression in N. tabacum, the corresponding coding sequence of 1276 bp was amplified from carrot storage root by RT-PCR with Pfu polimerase (Promega, Madison, WI, USA) and Psy2F and Psy2R primers (Supplementary Table S1) and cloned into pCR8 ® /GW/TOPO vector (Invitrogen ® , Waltham, MA, USA) according to the manufacturer's indications. Positive clones were selected by PCR, enzymatic digestion and sequencing (Macrogen Corp, Rockville, MD, USA, Supplemental Figure S1). Subsequently, pCR8/DcPSY2 positive clones were recombined into the binary vector pGWB2 [42], to generate pGWB2-DcPSY2, then introduced into Agrobacterium tumefaciens (GV3101 strain) for stable DcPSY2 expression in tobacco.

Plant-Material-and Agrobacterium-Mediated Transformation
Seeds of Nicotiana tabacum cultivar Xanthi NN were surface sterilized, plated in fullstrength Murashige and Skoog (MS) medium supplemented with 1% sucrose and solidified with 0.8% agar, then transferred to a growth room used for in vitro cultivation equipped with a 16 h light/8 h dark photoperiod (photon fluence rate of 100 µmol m −2 s −1 ) at Plants 2023, 12,1925 5 of 17 21-23 • C for 4 weeks for Agrobacterium-mediated transformation [43]. Antibiotic-resistant shoots regenerated in the presence of 5 mg/L BAP and 1 mg/L IBA were excised from initial explants and transferred to hormone-free MS medium containing 50-100 mg/L kanamycin and 300 mg/L cefotaxime. When seedlings reached about 7 cm in height, with proper root development, the obtained T 0 plants were transferred to plastic pots (20 × 10 cm) containing a mix of soil and vermiculite (2:1) and cultivated in a greenhouse under a 16 h long day photoperiod illuminated with cool white fluorescent light (115 µmol m −2 s −1 ) at 20-23 • C. NptII (kanamycin resistance gene) and DcPSY2 amplification were carried out to select T 0 tobacco transgenic lines (Supplementary Figure S4). Three T 0 transgenic lines (L3, L4 and L6) were selected for gene expression and carotenoid composition in 2-month-old transgenic plants. Then, around 200 seeds of L3, L4 and L6 transgenic T 0 lines were sown in MS-Suc − (Murashige and Skoog, 1962) supplemented with 100 mg/L kanamycin to select heterozygote and homozygote T 1 lines for further analysis.

RNA Extraction and Quantitative RT-PCR
Total RNA was extracted using a modified method based on [44]. Samples were collected from 17-day-old wild-type carrot seedlings, from carrots cultivated at 4, 8 and 12 weeks in the greenhouse, from leaves of 2-month-old T 0 tobacco transgenic plants and from entire roots and leaves of 1-month-old T 1 L3, T 1 L4 and T 1 L6 tobacco transgenic lines. Samples were immediately frozen in liquid N 2, then stored at −80 • C. Afterwards, tissue samples were homogenized in the presence of liquid N 2 and one aliquot (50-100 mg of powder) was incubated with 700 µL of extraction buffer containing 2% (w/v) CTAB, 2% (w/v) PVP40, EDTA 25 mM, NaCl 2M, Tris-HCl (pH 8.0) 100 mM and 0.05% spermidine trihydrochloride in DEPC-treated water. Samples were incubated at 65 • C for 5 min in 1000 rpm agitation. Thereafter, two chloroform:isoamylic alcohol (24:1) extractions were performed. Supernatant was precipitated with 1 volume isopropanol and 0.1 volumes of 3M sodium acetate (pH = 5.2) for 10 min at room temperature. Samples were centrifuged and RNA pellets were washed with 75% ethanol in DEPC-treated water. Subsequently, pellets were dried and resuspended in 30 µL of ultrapure DEPC-treated water and subjected to concentration, purity and integrity analysis. For cDNA synthesis, 1.0 µg of total DNA-free RNA was mixed with 0.6 mM of oligoAP primer (Supplemental Table S1) and Improm II reverse transcriptase (Promega ® , Madison, WI, USA). Quantitative RT-PCR (qRT) experiments were performed as described [45] in a Stratagene Mx3000P thermocycler, using SYBR Green double-strand DNA binding dye. Specific primers were designed to amplify a specific coding sequence from either carotenogenic genes: DcPSY1, (DQ192186), DcPSY2 (DQ192187), NtPSY1 (JF461341), NtPSY2 (JX101475), NtLCYB (KC484706), or saltresponsive genes: NtNCED3 (JX101472.1) and NtOsmotin (X61679.1). As normalizers, the gene expressions of RNAr18S (AJ236016.1) and NtEF1α (AF120093.1) were determined (Supplemental Table S1). Relative transcript levels of all genes of interest were obtained by introducing fluorescence results in the described equation [46]. Expression analysis was performed by means of three biological replicates and two technical repeats. In the case of T 1 L3, T 1 L4 and T 1 L6 transgenic lines, three pools of three plants each were used as the three biological replicates that were measured twice. In all cases, the reaction specificities were tested with melting gradient dissociation curves. To test for significant differences in gene expression, results were subjected to a two-tailed Student's t-test (p < 0.05, confidence interval 95%) according to the General Linear Models option in the statistical software package Graphpad Prism.

Carotenoid and Chlorophyll Extraction and High-Performance Liquid Chromatography (HPLC-DAD)
Pigments from 2-month-old T 0 DcPSY2 L3, L4 and L6 lines and control lines transformed with the pGWB2 empty vector (E/V) were obtained according to [45]. Carotenoids were extracted from 100 mg of pooled leaves with 1 mL of hexane:acetone:ethanol (2:1:1 v/v), then centrifuged. The upper phase was collected, then dried under a stream of N 2 and Plants 2023, 12,1925 6 of 17 resuspended in 1 mL of acetone. Total carotenoid quantification was carried out in a Shimadzu Spectrophotometer at 474 nm (absorption of carotenoids and chlorophyll), 645 nm (chlorophyll a) and 662 nm (chlorophyll b). Pigments were separated in a Shimadzu HPLC (LC-10AT) equipped with a diode array detector using an RP-18 Lichrocart 125-4 reverse phase column (Merck ® , Rahway, NJ, USA), and a mix of acetonitrile:methanol:isopropanol (85:10:5 v/v) as a mobile phase. Separation was performed with a 1.5 mL/min flow rate at room temperature under isocratic conditions. Results were analyzed with LCsolutions ® package software version number 1.0.0.1. For phytoene measurements, chromatograms at 285 nm were obtained. Carotenoids were identified according to their absorption spectra, retention time and comparison with specific pigment standards, which were corroborated by comparison with the Carotenoids Handbook [47,48]. All operations were carried out in triplicate, on ice and dark conditions, to avoid photodegradation, isomerization and structural changes of carotenoids.

Norflurazon Treatment
DcPSY2 T 1 L6 transgenic tobacco seedlings were grown in vitro in full-strength MS media without sucrose (MS-Suc − ) supplemented with 100 mg/L kanamycin for 14 days. Subsequently, half of the seedlings were placed in MS-Suc − media in the presence of 100 µM of the PDS inhibitor Norflurazon (NFZ) for 7 days [49], then frozen in liquid N 2 and stored at −80 • C until carotenoid extraction and phytoene quantification were achieved. This experiment was carried out with three biological replicates of 15 pooled T 1 L6 transgenic seedlings from control and herbicide treatment conditions.

Salt Stress Tolerance Assay
T 1 E/V, T 1 L3 and T 1 L6 transgenic lines were aseptically sown on agar plates containing MS-Suc − supplemented with 100 mg/L kanamycin. After 13 days of in vitro cultivation, different groups of antibiotic-resistant seedlings were placed on Petri dishes containing 25 mL of fresh MS-Suc − medium with or without 250 mM NaCl. After 16 days of the respective treatment, the fresh weight of individual lines was determined, before their transfer to soil for cultivation in greenhouse conditions (16 h light/8 h dark photoperiod, photon fluence rate of 100 µmol m −2 s −1 at 21-23 • C). Following 21 days of recovery, survival rates of each group of plants were calculated. This experiment was carried out in triplicate using between 10 and 30 T 1 E/V, T 1 L3, T 1 L4 and T 1 L6 transgenic T 1 E/V, T 1 L3, T 1 L4 and T 1 L6 transgenic seedlings for each treatment.

ABA Quantification
T 1 E/V, T 1 L3 and T 1 L6 transgenic seedlings cultivated for 21 days in full-strength MS medium supplemented with 1% sucrose were subsequently transferred to hydroponic medium and allowed to grow for an additional 10 days before acute salt treatment. An amount of 200 mg of leaves and root tissue obtained from hydroponically grown T 1 (n = 3) seedlings subjected to 250 mM NaCl during 0 and 8 h were flash frozen and ground with a mortar and a pestle in the presence of liquid N 2 and 4 mL of H 2 0 milli-Q. The mixture was shaken at 4 • for 20 min and incubated overnight at 4 • C. Afterwards, the samples were centrifuged at 14,000 rpm for 15 min, and the aqueous phase containing ABA was recovered. ABA determination was performed by the HPLC-ESI-MS/MS system (Agilent 1200 series, MS/MS 6410, Agilent Technologies, Santa Clara, CA, USA) following the instructions described in [50]. The mobile phase was composed of 0,1% formic acid. A sample of 20 µL was separated using a Zorbax C18 (4.6 × 150 mm, 5 µm) reverse phase column with a flow rate of 0.3 mL/min at room temperature. The experiment was carried out in triplicate with 3 plants each.

DcPSY2 In Silico Analysis and Expression during Carrot Development
The DcPSY1 (1197 bp) and DcPSY2 (1276 bp) coding sequences were obtained by conventional RT-PCR, cloned into pCR ® 8/GW/TOPO and sequenced (Supplementary Figure S1). The predicted proteins DcPSY1 (398 aa) and DcPSY2 (437 aa) share 64% identity and present the same amino acids at the active site, substrate binding pocket, Mg 2+ binding site and the squalene/phytoene synthase domains (Supplementary Figure S3), isoprenoid synthase, and trans-isoprenyl diphosphate synthase domains found in functional PSY enzymes (Figure 2A). A multiple alignment including AtPSY (AAA32836.1), NpPSY (CAA55391.1), DkPSY (ACM44688.1), ZmPSY (ACY70935.1), DcPSY1 (DQ192186.1), and DcPSY2 (DQ192187.1) showed that DcPSY2 presents the characteristic functional domains of the PSY enzymes such as Trans_IPPS-HH, the aspartate-rich regions, residues that cover the active site and the substrate-binding pocket (Supplementary Figure S4). Although the amino acid sequence is highly similar, the expression profile of both PSY paralogs is different in carrot. DcPSY1 presents a higher level of expression than DcPSY2 in leaves, especially at 4 and 8 weeks of development, when it presents 60-fold and 6-fold more expression than DcPSY2 ( Figure 2B). On the other hand, in roots, DcPSY2 presents 12-fold, 37-fold and 56-fold more expression than DcPSY1 at 4, 8 and 12 weeks of development, respectively ( Figure 2B).

Expression of DcPSY2 in N. tabacum Promotes an Increment in Carotenoid Content
Once the correct DcPSY2 sequence identity was confirmed, it was cloned and pGWB2-DcPSY2 (35SCaMV:DcPSY2) vector was obtained to transform tobacco leaf disc explants. Transformed plants were selected with kanamycin and genotyped by amplifying the transgene DcPSY2 (Supplementary Figure S5). Three lines were selected, as they presented the most similar phenotype with E/V plants. Our analysis of expression levels indicates that line L4 presents a 2.5-and 25-fold higher expression level than L3 and L6, respectively ( Figure 3A). Remarkably, the total carotenoid content in leaves of two-month-old transgenic T 0 tobacco lines were higher in lines L3 and L6 than in L4, which indeed presents a significant reduction. Particularly, line L3 had a 44% increase in β-carotene content, a 41% rise in total carotenoids and a 63% increase in α-carotene, whereas L6 had an increase of 57% in β-carotene and total carotenoid and 59% increased lutein content with respect to E/V lines ( Figure 3B). On the contrary, L4 did not present any difference in individual carotenoid composition. Interestingly, the chlorophyll level was also altered in transgenic plants. L3 presents an increment in chlorophyll a of 35% and L6 has an increase in chlorophyll b and chlorophyll a of 57% and 60%, respectively, with respect to E/V lines. In contrast to other results, L4 showed a reduction of 47% in both chlorophylls ( Figure 3C).
Looking deeper on the DcPSY2 function, the accumulation of phytoene in the T 1 generation of L6 was assessed. T 1 seedlings of L6 were selected because T 0 L6 presented the highest increment in carotenoids and chlorophylls. For this purpose, T 1 L6 seedlings grown on MS-agar plates for 14 days were transferred to fresh MS-agar containing the herbicide norflurazon (NFZ) which, via phytoene desaturase inhibition, leads to the accumulation of the colorless carotenoid phytoene. To ensure maximum inhibition of phytoene desaturase activity, a high concentration of NFZ was employed (100 µM; [49]). After 7 days of treatment, 35SCaMV:DcPSY2 T 1 L6 as well as the T 1 E/V control lines showed evident symptoms of bleaching (not shown). Quantitative HPLC analysis of carotenoids of the aerial tissue revealed that T 1 L6 seedlings showed a 40% increase in phytoene content after NFZ treatment with regard to T 1 E/V plants ( Figure 3D), thus demonstrating that phytoene is converted to downstream carotenoids in seedlings grown in vitro. To determine the effect on the endogenous carotenoid pathway, an analysis of the expression of tobacco carotenogenic genes was determined. Interestingly, NtPSY1 and NtPSY2 expression was impaired in transgenic lines, whilst NtLCYB expression was not affected in L3 and L6 or induced in L4 ( Figure 3E), suggesting that the increment in carotenoids and chlorophylls is due to the expression of DcPSY2. The results presented here add to the previous discovery made by Arias et al. (2021), demonstrating that DcPSY2 encodes an active enzyme that results in an increase in carotenoids and chlorophylls. Looking deeper on the DcPSY2 function, the accumulation of phytoene in the T1 generation of L6 was assessed. T1 seedlings of L6 were selected because T0 L6 presented the highest increment in carotenoids and chlorophylls. For this purpose, T1L6 seedlings grown on MS-agar plates for 14 days were transferred to fresh MS-agar containing the herbicide norflurazon (NFZ) which, via phytoene desaturase inhibition, leads to the accumulation of the colorless carotenoid phytoene. To ensure maximum inhibition of phytoene desaturase activity, a high concentration of NFZ was employed (100 µM; [49]). After 7 days of treatment, 35SCaMV:DcPSY2 T1L6 as well as the T1E/V control lines showed evident symptoms of bleaching (not shown). Quantitative HPLC analysis of carotenoids of the aerial tissue revealed that T1L6 seedlings showed a 40% increase in phytoene content after NFZ treatment with regard to T1E/V plants ( Figure 3D), thus demonstrating that phytoene is converted to downstream carotenoids in seedlings grown in vitro. To determine the effect on the endogenous carotenoid pathway, an analysis of the expression of Ubiquitin was used as housekeeping gene and E/V samples were used as calibrator and settled in 1. Error bars represent standard error of the mean (two technical replicates; asterisks indicate Student's t-test significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Transgenic Tobacco DcPSY2 Lines Showed Salt Stress Tolerance in a Chronic Salt Stress Assay
The functionality of DcPSY2 was also evaluated in terms of conferring salt stress tolerance. For this purpose, we selected 13-day-old T 1 E/V, T 1 L3 and T 1 L6 seedlings to be subjected to an in vitro chronic 250 mM NaCl treatment ( Figure 4A). After 16 days of salt stress, DcPSY2-expressing seedlings presented a significantly higher fresh weight than E/V plants ( Figure 4A,B). Furthermore, when considering the control group, T 1 L3 exhibited a comparable fresh weight to T 1 E/V, and T 1 L6 did not exhibit a significant difference in fresh weight when compared to seedlings that were not subjected to stress ( Figure 4B). After salt stress treatment, plantlets were transferred to soil and cultivated in the greenhouse, without stress. The survival rate after 21 days of recovery ( Figure 4C) showed that T 1 L3 and T 1 L6 transgenic plants had a 40% and 68% survival rate, respectively, which is remarkably greater than that observed for control plants (16%; Figure 4D), but T 1 L3 presents a similar phenotype to T 1 E/V, contrary to T 1 L6. Considering these results, T 1 L3 lines present a better performance under control and saline stress conditions. erance. For this purpose, we selected 13-day-old T1E/V, T1L3 and T1L6 seedlings to be subjected to an in vitro chronic 250 mM NaCl treatment ( Figure 4A). After 16 days of salt stress, DcPSY2-expressing seedlings presented a significantly higher fresh weight than E/V plants ( Figure 4A,B). Furthermore, when considering the control group, T1L3 exhibited a comparable fresh weight to T1E/V, and T1L6 did not exhibit a significant difference in fresh weight when compared to seedlings that were not subjected to stress ( Figure 4B). After salt stress treatment, plantlets were transferred to soil and cultivated in the greenhouse, without stress. The survival rate after 21 days of recovery ( Figure 4C) showed that T1L3 and T1L6 transgenic plants had a 40% and 68% survival rate, respectively, which is remarkably greater than that observed for control plants (16%; Figure 4D), but T1L3 presents a similar phenotype to T1E/V, contrary to T1L6. Considering these results, T1L3 lines present a better performance under control and saline stress conditions.

DcPSY2 Transgenic Plants Induce a Boost in ABA and Stress-Associated Genes under Acute Salt Treatment
To evaluate if ABA is synthesized when plants are subjected to salt stress, representative T 1 E/V, T 1 L3 and T 1 L6 transgenic seedlings were exposed to 250 mM NaCl for 8 h. We found that T 1 L3 and T 1 L6 presented a 1.6-and 3-fold raise in ABA levels in leaves, respectively, after NaCl treatment, with respect to unstressed plants (C), which is significantly lower than the 4-fold increase observed in T 1 E/V control plants under similar conditions ( Figure 5A). However, T 1 L3 and T 1 L6 present 1.3-and 2.5-fold increases of ABA, respectively, in roots, with respect to unstressed plants (C), whereas T 1 E/V transgenic plants produced no significant changes in ABA levels ( Figure 5A). This finding implies that the improved salt resistance in DcPSY2 plants may be linked to a heightened ABA response in the roots at an early stage. To determine whether the increase in ABA in the DcPSY2-expressing transgenic plants was associated with an enhanced expression of salt stress-related genes, we evaluated the transcript abundance of genes that encode enzymes related to ABA production such as the 9-cis-epoxycarotenoid dioxygenase 3 (NtNCED3) and the osmotic stress response gene, NtOsmotin [51]. After 2 h of exposure to salt, there was a 15-fold increase in NtNCED3 transcript levels in T 1 E/V lines in above-ground tissue, which then decreased to lower levels after 4 h ( Figure 4B). Notably, transgenic DcPSY2 lines showed a progressive increase in NtNCED3 transcript levels at 2 h and 4 h of salt exposure. Particularly, T 1 L3 presented 4.7-to 7.7-fold increases at 2 h and 4 h, respectively; similar to T 1 L6, with 2.2-and 5.3-fold increases in NtNCED3 transcript abundance at 2 h and 4 h, respectively ( Figure 5B). Interestingly, NtNCED3 presents a higher basal level of expression in T 1 L3 and T 1 L6 than in T 1 E/V plants (it is due to this that the increment in T 1 L3 and T 1 L6 is lower than that in T 1 E/V at 2 h) ( Figure 5B). The relative transcript level of NtNCED3 in roots increased by approximately 49 times in T 1 E/V, but after 4 h of salt treatment, it decreased and showed no significant differences ( Figure 5B). In contrast, transgenic DcPSY2 lines showed a sustained increase even at 4 h of salt treatment, as observed for T 1 L3 (15-and 29.2-fold increases after 2 h and 4 h, respectively) and T 1 L6 (81.6-and 69.7-fold increases after 2 h and 4 h, respectively) ( Figure 5B). In the case of NtOsmotin, the transcript levels in aerial tissue remained unaltered between 2 h and 4 h of salt treatment in T 1 E/V, as in T 1 L3, but increased about 7 times in T 1 L6 at 4 h of NaCl treatment ( Figure 5C). However, most interestingly, and in correlation with ABA levels ( Figure 5A), NtOsmotin transcript levels increased in roots inT 1 L3 and T 1 L6 after 4 h of salt treatment (1.9-and 2.6-fold increases, respectively), whereas T 1 E/V showed no significant changes after stress application ( Figure 5C). It is important to consider that NtOsmotin presents an upper basal level of expression in T 1 L3 and T 1 L6, but not in T 1 E/V plants. Collectively, the T 1 L3 and T 1 L6 transgenic lines exhibited increased and prolonged transcript levels of NtNCED and NtOsmotin, which corresponded with the levels of ABA in both leaves and roots during the exposure-to-salt-stress treatment.

Endogenous NtPSY Gene Expression Is Enhanced in DcPSY2 Transgenic Lines after Salt Stress Treatment
Some PSYs are described to be upregulated upon salt stress, acting as an indicator of salt stress exposure and response [11,29]. Hence, we assessed the transcript levels of NtPSY1 and NtPSY2 in the above-ground and underground tissues at 0 h, 2 h and 4 h of exposure to 250 mM NaCl. This analysis aimed to explore how the expression of DcPSY2 affects the regulation of these endogenous genes during salt stress conditions. In leaves, NtPSY1 transcript levels remained unchanged in T 1 E/V control lines but increased 2.7-fold in T 1 L3 and T 1 L6 at 2 h of salt treatment, and then dropped to normal levels at 4 h ( Figure 6A). In roots, NtPSY1 transcript levels increased 3-fold after 2 h in T 1 E/V, whereas a 3.5 and 4.7-fold increase was registered in T 1 L3 and T 1 L6, respectively. At 4 h of salt treatment, NtPSY1 transcript levels dropped to no significant amounts with respect to time 0 in T 1 E/V and T 1 L3, in contrast with T 1 L6, which showed a sustained 3.1-fold increase of these transcript levels ( Figure 6A). In the case of NtPSY2, it peaked at 2 h of salt stress treatment in leaves, then dropped to basal levels at 4 h in T 1 E/V, but showed a permanent induction for both T 1 L3 and T 1 L6 even at 4 h of treatment. For T 1 L3, there was a 3.0-fold induction at 2 and 4 h of salt stress, whilst T 1 L6 showed a 3.7-to 6.3-fold increase in NtPSY2 relative to transcript levels at 2 h and 4 h of treatment ( Figure 6B). In roots, we found a 4.4-fold increase in induction after 2 h of salt stress in T 1 E/V control lines and 5.6-and 3.7-fold increases in T 1 L3 and T 1 L6 under similar conditions. Moreover, for both T 1 E/V and T 1 L3, NtPSY2 transcript levels decayed to basal values at 4 h but retained a 4.1-fold increase in induction in T 1 L6 ( Figure 6B). The findings suggest that salt stress triggers the upregulation of NtPSY1 and NtPSY2 genes in both tobacco T 1 E/V transgenic lines and those expressing DcPSY2. However, T 1 L3 and T 1 L6 lines exhibited a sustained increase in transcript accumulation, which may contribute to enhancing their salt stress tolerance. bars represent standard error of the mean. Asterisks denote significant differences by twounpaired Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001).

Endogenous NtPSY Gene Expression is Enhanced in DcPSY2 Transgenic Lines after S Stress Treatment
Some PSYs are described to be upregulated upon salt stress, acting as an indica salt stress exposure and response [11,29]. Hence, we assessed the transcript leve NtPSY1 and NtPSY2 in the above-ground and underground tissues at 0 h, 2 h and 4 exposure to 250 mM NaCl. This analysis aimed to explore how the expression of Dc affects the regulation of these endogenous genes during salt stress conditions. In le NtPSY1 transcript levels remained unchanged in T1E/V control lines but increased 2.7 Three biological replicates, each composed of three plants, were performed and real-time RT-PCR was measured in triplicate. EF1 alpha was used as housekeeping gene and each E/V 0h (control) was used as calibrator and settled in 1. Error bars represent standard error of the mean. Asterisks denote significant differences by two-tailed unpaired Student's t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001). ns denote no significant differences.
NtPSY2 transcript levels decayed to basal values at 4 h but retained a 4.1-fold increase in induction in T1L6 ( Figure 6B). The findings suggest that salt stress triggers the upregulation of NtPSY1 and NtPSY2 genes in both tobacco T1E/V transgenic lines and those expressing DcPSY2. However, T1L3 and T1L6 lines exhibited a sustained increase in transcript accumulation, which may contribute to enhancing their salt stress tolerance. Three biological replicates, each composed of three T1 plants, were performed, and real-time RT-PCR was measured in triplicate. EF1 alpha expression was used as normalizer and each E/V 0 h (control) was used as calibrator and settled in 1. Error bars represent standard error of the mean. Asterisks denote significant differences by two-tailed unpaired Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001).

DcPSY2 Boost Carotenoid, Phytoene and Chlorophyll Content in N. tabacum
In silico analysis showed that DcPSY2 presents the characteristic functional domains of PSY enzymes (Figure 2, Supplementary Figure S4), suggesting its functionality. This was confirmed by the constitutive expression of DcPSY2, which led to up to a 1.4-fold increase in total carotenoids and a 1.5-fold increase of β-carotene in transgenic tobacco plants, indicating that DcPSY2 is functional in this heterologous model. Previously, we obtained a similar-fold induction in DcPSY2 transgenic tomato fruits, which produce a 1.2-to 1.3-fold increase in total carotenoid and up to a 1.25-fold increase in lycopene [52]. Subcellular localization [52] and phytoene accumulation ( Figure 3D) in lines subjected to NFZ treatment is another factor that supports the functionality of DcPSY2. Previous studies have indicated that carotenoid accumulation in photosynthetic tissues is strongly replicates, each composed of three T 1 plants, were performed, and real-time RT-PCR was measured in triplicate. EF1 alpha expression was used as normalizer and each E/V 0 h (control) was used as calibrator and settled in 1. Error bars represent standard error of the mean. Asterisks denote significant differences by two-tailed unpaired Student's t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001). ns denote no significant differences.

DcPSY2 Boost Carotenoid, Phytoene and Chlorophyll Content in N. tabacum
In silico analysis showed that DcPSY2 presents the characteristic functional domains of PSY enzymes (Figure 2, Supplementary Figure S4), suggesting its functionality. This was confirmed by the constitutive expression of DcPSY2, which led to up to a 1.4-fold increase in total carotenoids and a 1.5-fold increase of β-carotene in transgenic tobacco plants, indicating that DcPSY2 is functional in this heterologous model. Previously, we obtained a similar-fold induction in DcPSY2 transgenic tomato fruits, which produce a 1.2-to 1.3-fold increase in total carotenoid and up to a 1.25-fold increase in lycopene [52]. Subcellular localization [52] and phytoene accumulation ( Figure 3D) in lines subjected to NFZ treatment is another factor that supports the functionality of DcPSY2. Previous studies have indicated that carotenoid accumulation in photosynthetic tissues is strongly dependent on and co-regulated with chlorophyll biosynthesis and accumulation [53]. Consistent with that study, DcPSY2 tobacco lines also showed greater chlorophyll levels, especially chlorophyll a ( Figure 3C).
Total carotenoid and chlorophyll a content correlated inversely with the relative transcript abundance of DcPSY2 transgene in each line. To be more specific, the expression levels of DcPSY2 that are categorized as low (L3) and moderate (L6) induced significant enhancements in carotenoid and chlorophyll content. On the other hand, the highest expression level (L4) either did not cause any changes or caused a decrease in these levels ( Figure 3A-C). This phenomenon has been observed in other studies and species [28,54], demonstrating the intimate association between PSY expression level and the regulation of carotenoid accumulation. As an explanation for this phenotype, we hypothesize that low or moderate DcPSY2 expression leads to an increased carotenoid content through a retrogradecrosstalk mechanism with chlorophyll biosynthesis [55], and that an increment of transgene abundance over a threshold level induces an opposite effect on carotenoid accumulation. This observation was previously reported [28] after expression of NtPSY genes in tobacco plants. It is important to remember that inserting a foreign gene into a plant genome is always random, and it may also affect other genes, resulting in gene silencing or altered expression. Furthermore, it is also possible that high expression of NtPSY2 may associate with lower levels of gibberellins and chlorophylls, since it is reported that PSY uses GGPP as a substrate, which is also the precursor for the synthesis of chlorophylls as well as gibberellins [40,55,56] Transcript levels of endogenous NtPSY1 and NtPSY2 were significantly reduced in DcPSY2 transgenic lines and showed unaltered or decreased NtLCYB transcript levels ( Figure 3E) in accordance with previous studies carried out on the expression of DcLCYB1 gene in carrot and tobacco [45,55]. In relation to these findings, we hypothesize that the reduction in the endogenous NtPSY transcript levels could be linked to a mechanism targeted to diminishing the metabolic flux towards the carotenogenic pathway, in order to maintain other metabolic routes requiring the common precursor, GGPP, such as the chlorophyll pathway (Figure 1). In accordance with this proposal, authors reported that the expression of DcLCYB1 in tobacco showed an increment in carotenoid, chlorophyll and gibberellin (GA) through positive feedback on the expression of metabolic precursor genes, such as DXS and GGPPS, among others [55]. In our work, we noticed that T 1 L6 are smaller in size than T 1 L3 and T 1 E/V plants, even in the absence of salt (Figure 4) during the first month of culture, which could be associated with a reduction in gibberellin levels. These results were comparable to those reported by Fray et al., (1995) from expressing the fruit paralog SlPSY in tomato, which resulted in a dwarf phenotype that was attributed to a reduction in GA levels and a slight increase in ABA levels due to competition for the GGPP substrate [28,56]. Additionally, the high NtPSY transcript abundance in tobacco transgenic plants produced stunted plants with a decrease in total pigment content, along with phytoene accumulation [28]. However, upon reaching adulthood, T 1 L6 DcPSY2 plants reached a phenotype similar to T 1 L3 and T 1 EVs, suggesting that their development time is only slower. Together, these results indicate that the increased carotenoid and chlorophyll content in DcPSY2-expressing lines was a direct consequence of the level in transgene expression.

Constitutive Expression of DcPSY2 Leads to an Increase in Salt Tolerance and ABA Levels in Transgenic Tobacco
Climate change produces water deficit and salinity in soils, impairing growth and production in plants [57]. Plants have developed mechanisms to cope with stressful situations [58]. Among them, plants activate tolerance through ABA-dependent and independent mechanisms [16]. ABA is synthesized through the carotenoid pathway, being PSY and NCED key limiting steps in those syntheses. Previous reports indicate that ectopic expression of PSY genes promotes salt tolerance in transgenic organisms [34,59,60]. Similarly, we observed that the constitutive expression of DcPSY2 transgene enhanced salt tolerance, coupled with higher levels of NtNCED expression and ABA level, preferably in roots. Salt tolerance in terms of fresh weight and survival rate after the saline treatment was in direct correlation with DcPSY2 transcript levels. Even though the survival rate of T 1 L6 plants was better, this did not lead to better growth, which means that T 1 L3 plants might have a higher yield also when the plants are older ( Figure 4B,C). The boosted fresh-weight performance under saline stress in transgenic DcPSY2 plants may also be attributable to reduced water loss under salinity conditions [61,62] and to the significant increment in ABA level [34,63]. Salt stress treatment for 5 h induced ABA accumulation in shoots and roots of Arabidopsis wild-type plants that was accompanied by an increment in carotenoids and expression of PSY in the root [34]. Previously, we determined that DcPSY2 and DcPSY1 are induced by salt stress, but only DcPSY2 by ABA, in which ABRE transcription factors mediate the expression of DcPSY2 [40]. These results complement the previous findings, suggesting that DcPSY2 confers salt stress tolerance when overexpressed in plants. The functional role of DcPSY1 in this process remains to be determined.
The increase in transcript abundance of NtNCED3 correlates with ABA levels, considering that the expression of NCED is required for ABA synthesis. Interestingly, NtNCED presented 4-to 6-fold higher basal levels of expression in leaves of L3 and L6 with respect to E/V plants, and presented a sustained induction at 2 h and 4 h in leaves and roots after acute NaCl treatment ( Figure 5B). Although in E/V, NtNECED was induced only at 2 h, it revealed that the acute stress assay was correctly achieved. Osmotin, a later gene in the ABA stress response pathway, was induced preferably in roots of transgenic lines at 4 h ( Figure 5C), whereas E/V plants did not show any induction, suggesting that in control plants, the response is delayed.
We propose that the expression of DcPSY2 in tobacco leads to an increment in the NtNCED3 expression level, which permits the production of ABA preferably in the root, leading further to the induction of NtOsmotin in the root. This is also sustained by the fact that under salt stress, endogenous NtPSY is also upregulated ( Figure 6) to permit enough metabolite supply for stress tolerance and for ABA synthesis.

Conclusions
Taken together, these results lead to the conclusion that DcPSY2 codifies for a functional enzyme that enhances carotenoid and chlorophyll content in transgenic tobacco lines, and promotes salt stress tolerance in tobacco through an enhancement of the ABA level as well as the expression of genes involved in abiotic stress tolerance. The enhanced salt stress tolerance exhibited by DcPSY2 transgenic plants could serve as a valuable biotechnological strategy for rootstocks that have significant economic importance. This is especially relevant because rootstocks are in direct contact with the saline stress present in the soil.